Ch. "40" - Immunological techniques Flashcards
Immunoassay
Using antigens and antibody complexes to generate measurable results.
Blocking
Blocking: a protein in which the primary or secondary antibodies don’t bind to, blocks open spots, most common: BSA (Brumine serum albumin), fish gellatin and dry milk powder, essential to keep the specificity of the mechanism.
Primary and Secondary antibodies
- The primary antibody is specific for the antigen of interest
- The secondary antibody is bound to the primary through the constant region of the primary antibody, therefore you can use the same secondary antibody for multiple different primary antibodies, cost efficient
- The secondary Ab can be used for all primary Abs of the same subclass produced in the same species
- Secondary Abs are normally labeled for detection (enzyme: AlkalinePhosphatase/peroxidase, fluorescent, GFP, or just biotin/streptavidin)
Western blotting
Detection technique in which a probe, usually an antibody, binds to a protein target molecule. Does not involve nucleic acid hybridization. Proteins are separated on a gel, transferred to a membrane, and detected by their corresponding antibodies.
A primary antibody specific for the protein binds to the protein. A labeled, general secondary antibody is used to detect the proteins. Used to detect polypeptide, size and structure can somewhat be expected.
Western blotting:
- To detect proteins of interest, and to semi-quantify the concentration of them
- Start by gel electrophoresis, SDS-PAGE
- The proteins are negatively charged by the SDS, and the polyacrylamide gel electropphoresis (PAGE) seperated the proteins based on molecular mass
- The proteins with low molecular mass will migrate further, the approximately molecular mass of the protein of interest can then be determined by comparison to a known ladder
- The proteins are then transferred to a nitrocellulose or nylon membrane by electrotransfer, electric current
- The open spots are blocked, and primary Abs are added
- Primary antibodies bind to the proteins of interest, several antibodies per protein
- Wash, the primary antibodies not bound are removed
- Secondary Abs are added, and these bind to the primary antibodies
- The secondary Abs are labeled with enzymes
- Substrates are added, and the enzymes makes colorful products out of them
- The approximate molecular mass of the protein of interest can be visualized/determined, and it gives some indication of the concentration of the protein of interest
ELISA
ELISA: Enzyme-Linked ImmunoSorbent Assay
- Concentration of antigens in cells and tissues
- Used on liquid samples, 96 well plate
- E.g. Protein mix including your protein of interest
- Sample is added to the well, and the proteins bind to the walls of the well, both the proteins of interest and others
- Leftover solution is removed, the wells are “washed” and the open spots in between are blocked
- Primary antibody solution is added, and these bind to the protein of interest
- Wash, solution and protein not bound is removed
- Secondary antibody solution is added, and these bind to the primary antibodies
- The secondary antibodies are labeled, most often with enzymes like AP/peroxidase
- Substrates are added, and the AP/peroxidase transforms the colorless substrate to a colored product
- The wells containing the protein of interest can be detected, also the level of color can be used to measure the concentration of the protein of interest
- Used to detect molecules that you don’t know what it its
- Negative sides of this technique: you cannot be sure that the color signal derives from your protein of interest
- The ELISA method can be modified e.g. It can detect antibodies by turning the method described around: using antigens to detect antibodies, PCR
Sandwich ELISA
Sandwich ELISA:
- Two primary antibodies detecting two epitopes on the protein of interest, secondary antibodies bind to these, same as for the other techniques
- Epitopes are between 5 AA and larger
- Epitopes don’t need to be linear along the protein, can be from different closely located regions of the protein
- The secondary, third and quaternary structure effects and determines the structure of the epitopes, and therefore which antibodies can bind to it
- If two antibodies compete on the same epitope, one can replace the other, if so, and the opposite is not true, its unidirectional
Immunocytochemical localization
Immunocytochemical localization:
- Localization of an antigen in a cell or tissue
- Antigen protein of interest on a membrane
- Primary antibodies are added to bind to the antigens
- Secondary antibodies are added to bind to the primary ones
- The secondary antibodies either produce a colored product from a substrate, or are bound to tags like fluorescence
- A microscope can be used to detect
- Used to detect a section of a tissue, micrometer thin, something inside a cell, or to locate the target antigen
Immuno-affinity chromatography
Immuno-affinity chromatography:
- Column with matrix, sphere of matrix is bound to antibodies
- Add solution containing the protein of interest to the column
- The protein of interest (antigen) will bind to the specific antibodies in the column matrix
- Wash buffer is added to the column to elute the proteins not binding to the antibodies, the column is then cleaned for all the other proteins in the sample solution
To elute the proteins, elution buffer with low pH is added
- Used for purification of proteins and ligands
Antibodies
Y-shaped proteins and receptors that bind to specific antigens. Primary antibodies bind to specific antigens. Secondary antibodies are more general and bind to primary antibodies.
Bottom part of antibody is called the Constant region, which contains carbohydrates (glycosylation).
Top part is the Variable part, which has a heavy chain and a light chain. At the ends of the top parts are two identical paratopes, hypervariable regions that bind epitopes on the antigen-protein.
Antibodies are often bound to each other in complexes. However, IGM and IGG are singular antibodies. Different classes of antibodies have different structures and specificity. Some antibody classes include IgM, secretory form of IgM, IgG, IgD, IgA, and IgE. The smallest antibody consists of four polypeptide chains.
Epitope
Part of the antigen protein that the antibodies recognize and bind to.
Immune cells:
DC: Dendritic cells, one type of antigen presenting cells (APCs), present antigens to other cells.
Macrophages: Eat and digest invading, foreign cells.
Monocytes: Precursors for macrophages.
Neutrophiles: Also eat invading, foreign cells, but fewer, and are then degraded. Signals other cells, like DCs and macrophages, that it “needs help”.
T-cells/T-lymphocytes: Developed from stem cells in the bone marrow.
TH: T-helper cells, help Tc develop, and stimulate B-cells to produce antibodies.
TC: T-cytotoxic cells, T8, sense invading cells and kill them.
T-cell receptors are always bound to the surface of the T-cells, and are released to the serum like B-cell receptors.
B-cells/B-lymphocytes: Developed from stem cells in the bone marrow. Produce Y-shaped proteins/receptors called antibodies, such as IGM and IGG. which bind to specific antigens. This way, antigens can be recognized and attacked. Only mature B-cells, also called plasma cells, release the receptors/antibodies from their surface. Antibodies are receptors that bind through their epitopes, to free antigens. Antigens are then attacked before they manage to invade cells. If the antigens bind to antibodies on the B-cell, the receptor-antigen complex is endocytosed by the B-cell.
Each of the T- and B-cells have specific receptors.
When infected: granulocytes signals macrophages, macrophages migrate to the lymph nodes and activate B-cells. B-cells are matured to plasma cells, and surface antibodies are released
One B-cell produce only one class of antibodies
T-cells must be activated by the same antigen as the B-cell
Monoclonal antibodies
Monoclonal antibodies: antigen has only one epitope for one antibody, the antibody recognizes and binds to only one epitope on the antigen.
Monoclonal antibodies are made in small organisms, mainly produced in cell cultures.
- Immunize e.g. A mouse, by injection of protein of interest
- Boost 2-3 times
- Kill mouse and take out the spleen
- The spleen is filled of matured B-cells, plasma cells, some of them produce Abs against protein of interest
- The plasma cells are fused with B-cell cancer cells to yield hybridomma cell
- Each Hybridomma cell is immortal and produces only one type of antibody, the monoclonal antibody
- Each will produce different antibody from each other
Polyclonal antibodies
Polyclonal antibodies: Can recognize and bind to multiple different epitopes of a single antigen. Cover many of the epitopes on the protein.
- Many different types of antibodies will bind to different epitopes on the protein, can cover variabilities such as mutations
- High dilution causes only the antibodies with the strongest affinity to the protein to bind, prevents cross-binding of the many antibodies
Polyclonal produced from blood of an animal (bigger organisms), the blood cells are removed and the serum Ig fraction is used. Usually made by several different antibody secreting plasma cell lineages.
Crash course polyclonal Abs:
- Immunize e.g. a rabbit by injection of protein of interest and adjuvants to enhance the immune response
- After some weeks, more shots are given, boosts
- Blood is harvested from the animal
- Blood cells, hydrogen and others are removed, leaving only the serum
- The Ig fraction is isolated from the serum, can seperate between classes, but not subclasses of antibodies
- There are thousands of different antibodies in the serum
Immune system and Vaccines
In humans, the immune system has two main branches:
- Innate: the initial immune system
- Adaptive: derived depending on components or antigens we are presented to
When we are infected, immune responses cause an inflammatory response: swelling, redness, heat.
Lymphatic liquid: from afferent to efferent
Transport antibodies and some matured plasma cells
Lymph nodes swell when infected, lots of cells
Inflammation, cytokines released
Humans have blood and lymph systems that are connected at lymph nodes.
Major lymphatic tissues:
- Bone marrow, legs, hips, blood cell production
- Peyer´s patches in the small intestine, digestive system, antigens from food
- Spleen, B-cells maturation
- Tonsiles
- Timus, T-cell maturation
- Lymph nodes, B-cell maturation
Vaccines: Principle is to introduce the pathogen or parts of it to initiate an immune response. This will activate memory cells so that the next time a cell is infected by this pathogen, it recognizes it and induces an immune response immediately. Sometimes a boost shot is needed.
Adjuvants
Components used in a vaccine to help induce the immune response. Can be several different compounds, such as oil, virus particles, dead/attenuated pathogens, lipid-molecules, or cytokines. Adjuvants can have negative effects.
