IHC 1-HT Flashcards
Immunohistochmistry at the HT level
Many pathologists prefer an IHC stain for H. pylori, because it is easy to see the brown chromogen (DAB) on the light blue (Mayer hematoxylin) background. Is this a Steiner stain?
No. It is common for pathologists to ask for a Steiner but mean the IHC stain. Technically, a Steiner & Steiner is a silver stain (black bacteria on a yellow-brown background) as opposed to the IHC stain (brown bacteria on a blue background).
What is an antigen?
Short for antibody generator, an antigen is any substance that causes the body to form an immune respone against it. They are usually proteins or sugars found on the outside of a threatening agent. Take all of the pathogens from outside of the body, add toxins and the body’s own cells; and you have a list of possible antigens.
What is an antibody?
Antibodies are glycoproteins produced by plasma cells as an immune response against antigens.
What are the y-shaped things traveling through this blood vessel? Why are they y-shaped?
They are antibodies. Antibodies have a y-shaped structure and can bind to almost any non-self surface.
True or False: Each antibody recognizes a specific antigen.
True. In fact, a specific epitope on that antigen, as antigens may have several epitopes. That is the “specificity” of an antibody for an antigen, and the reason they can target pathogens and not harm other cells.
What is another name for antibody?
Immunoglobulin.
True or False: Each B (or T) cell is specific to 1 antigen.
True. Each lymphocyte binds to 1 particular epitope on an antigen. There may be many BCRs (B cell receptors) or TCRs (T cell receptors) on each cell, but they all recognize the same epitope on an antigen.
What is a cytokine?
They are proteins released by immune cells to signal other immune cells. Basically a messaging system between immune cells.
What is a B cell receptor?
An antibody attached to the surface of a B cell rather than secreted into the blood stream.
True or False: All B cell receptors on a particular B cell are the same.
True. All the B cell receptors on that cell are identical.
How many protein chains are in an antibody?
An antibody consists of 4 polypeptide chains. 2 light chains and 2 heavy chains. They are called light and heavy because of their molecular weight.
True or False: The heavy chain is the INSIDE part of the Y on an antibody.
True.
Is the Kappa and Lambda part the heavy chain or the light chain?
Light chain.
Can plasma cells be cancerous?
Yes, myeloma is cancer of plasma cells. Plasma cell neoplasms can be benign or malignant. Both heavy and light chains are tested for myeloma.
IgA, IgD, IgE, IgG, IgM are all different classes of antibodies (immunoglobulins). What determines the class?
The heavy chain of the antibody.
How many antigen binding sites are on an antibody?
- There are 2 identical arms that can bind to 2 epitopes and cause pathogens to clump together (agglutinate), which assists in phagocytosis.
What is the most predominant class, or isotype, of immunoglobulin (antibody) found in the human body?
IgG makes up 75% of serum antibodies in humans.
What is an epitope?
The binding site on an antigen.
What is a paratope?
The binding site on an antibody.
Is there more than 1 paratope on an antibody?
Yes, there are 2 paratopes or binding sites on each antibody.
Is there more than 1 epitope on an antigen?
Yes, the surface of a typical antigen can have 100 or more epitopes.
Do both plasma cells (plasmacytes) and memory B cells clone?
Yes. The memory B cells, which have cell surface receptors (immunoglobulins), clone; and the plasma cells which secrete antibodies (immunoglobulins) clone. This is how so many antibodies are rapidly produced against one antigen.
What is an antigen with an antibody attached to it called?
An antigen-antibody complex.
True or False: Each set of plasma cell clones produces multiple identical antibodies.
True. Each plasma cell in each clone can produce multiple antibodies and all of the antibodies from all of the cells in that clone are identical.
How many antibodies can a single plasma cell produce?
Hundreds, sometimes thousands, per second.
In Immunology, is cloning about cells or antibodies?
Both. When B lymphocytes differetiate into memory cells and plasma cells, they clone. When those plasma cells produce identical antibodies, that is called antibody cloning.
What does BCR stand for?
B cell receptor, as opposed to T cell receptor. Both are cell surface receptors, also called immunoglobulins.
True or False: Antibodies secreted from plasma cells that are from the same clone are called monoclonal.
True.
What are antibodies against the same antigen but from different clones called?
Polyclonal. 1 antigen can have several different types of epitopes. Each type of epitope will fit a different antibody clone.
True or False: The duration of memory a B or T cells has for a specific antigen can vary.
True. The memory lasts a different length of time for each antigen. That’s why you sometimes need to get revaxed or boostered.
What is antiserum?
Blood serum containing antigen specific antibodies.
What is immunohistochemistry?
With IHC, antibodies are used to visualize antigens, especially in tumors, that could not otherwise be seen with th light microscope.
How are polyclonal antibodies produced?
As in vaccine development, animals are injected ( or immunized) with antigens, then blood is removed from the animal and the antiserum (blood serum containing antibodies) is separated and a heterogenous mixture of antibodies isolated and collected.
How is a homogenous mixture of monoclonal antibodies produced.
Once the injected animal has produced antibodies to an antigen, the spleen is removed and specific B cell clones are isolated. Clones (against 1 epitope on the antigen) are fused with a myeloma cell line to create a hybridoma, which will continue to grow the monclonal antibodies.
What is myeloma?
Cancer that originates in plasma cells. These immortal cells are used in monoclonal antibody production.
What is a hybridoma?
The fusion (chemically or electrically) of epitope specific B cells with myeloma cells to produce monoclonal antibodies.
So, what is the difference between polyclonal and monoclonal antibodies again?
Polyclonal antibodies are SENSITIVE heterogenous (multiple epitopes on an antigen) and usually made in rabbits. Monoclonal antibodies are SPECIFIC homogenous (1 epitope on an antibody) and usually made in mice.
Why do we need an antigen or epitope retrieval step?
Also called unmasking. The bonds formed with formalin fixation can mask, or hide, antigen binding sites (epitopes).
Why do we need blocking steps?
Because background staining can obscure visualization of the targeted antigen or cause false positive results.
What causes background staining?
Nonspecific antibody binding, which can happen especially with polyclonal antibodies (due to multiple epitope binding), and endogenous enzyme activity (usually with hematopoietic tissue-like bone marrow).
True or False: The step that determines the IHC method is the Antibody Application step.
True. Direct, Indirect, Bridge, and Immune-Complex Methods differ in how antibodies are applied.
What is the difference between the Direct Method and the Indirect Method of IHC staining?
Direct-primary antibody is conjugated to a label in one step. Used in Immunofluorescence. Indirect-there is a secondary antibody bound to the primary antibody, amplifying the signal and making it more sensitive.
What is the Bridge Method of antibody application? Is it direct or indirect?
Indirect. A secondary antibody bridges between a primary and tertiary antibody of the same species. This also amplifies the signal.
What is the ABC Staining Method.
An indirect method using an Avidin/Biotin Complex to amplify the signal. However, endogenous biotin must be blocked with avidin when using this method.
What is the Dextran Strand Polymer Immune Complex Staining Method?
With this indirect method, primary and secondary antibodies are conjugated to a polymer chain that provides multiple binding sites. It is extremely specific and sensitive. It is the most popular method used.
Indirect IHC methods use more than a single (primary) antibody (i.e. secondary, tertiary, etc.) How are these antibodies made?
Secondary antibodies bind to the primary antibody INSTEAD of binding to the target antigen. To acheive this, the host species used to raise the secondary antibody must be different than the primary antibody host species. This extends to the next antibody, if there is one. The prefix “anti” is added to antibodies to describe what they are acting as antigens against.
True or False: The secondary antibody host species in an indirect method must match the primary host species. For example: If the primary antibody is made in a mouse, the secondary antibody must be made in a mouse.
Absolutely False. Opposites attract. You are forming another antigen-antibody complex. In order for the primary antibody to act as an antigen for the secondary antibody to link to, the animal species must be DIFFERENT and against the primary antibody species (example in this case: goat anti-mouse).
True or False: If the primary antibody was made in a rabbit, then the secondary antibody must be made against rabbit (anti-rabbit).
True. The secondary antibody could be goat anti-rabbit.
Does the isotype (class) matter when choosing secondary antibodies?
Yes, secondary antibodies should be raised against the host species including the isotype (class) of the primary antibody. So, if the primary antibody was raised in a rabbit and the isotype was IgG, they the secondary antibody could be Goat IgG anti-rabbit IgG.
True or False: In general, IHC staining areas can be divided into nuclear, cytoplasmic, and membranous.
True.
What is the detection step in IHC staining?
The step where a chromagen and substrate are applied. It is the step that allows visualization of the target antigen with a light microscope.
What is different about Immunofluorescense?
The antibody is conjugated with a fluorescent tag, must be viewed with a fluorescent scope, and it will fade with time.
What is the most common chromogen used in enzyme immunohistochemistry?
DAB. 3-3’ diaminobenzidine. It is used with the enzyme horseradish peroxidase and the substrate hydrogen peroxide to give a brown precipitate.
DAB is the most common chromogen used in IHC. Its enzyme is horseradish peroxidase, and, therefore, it must be blocked with hydrogen peroxide. What other chromogen uses this enzyme and blocking reagent?
AEC. 3-amino-9-ethycarbazole. It is a red precipitate that requires aqueous coverslipping.
In addition to unmasking epitopes, what is an advantage to epitope retreival?
You can dilute expensive antibodies and use less of them.
What are the two methods of epitope (also called antigen) retrieval?
EIER and HIER. Enzyme induced epitope retrieval and heat induced epitope retrieval.
Name some enzymes used in EIER.
Trypsin, pepsin, pronase, protease.
Name some buffers used in HIER.
PBS, zinc sulfate, citrate, EDTA
What is the only antibody isotype that is small enough to cross the placental barrier.
IgG. These can last a lifetime, but take 7-14 days to produce on the 1st encounter. This is the isotype we are trying to produce when we get vaccinated.
What is the antibody isotype with an unknown function?
IgD
Which antibody isotype has a dimer structure?
IgA. It is found in saliva and mucus.
Which antibody isotype protects against parasites?
IgE. Also allergic reactions. Combines with mast cells to release histamine.
Which antibody isotype has a pentamer structure?
IgM
How many antibody isotypes are there?
- IgA, IgD, IgE, IgG, IgM
Which antibody isotype is found on mast cells?
IgE. Deals with pollen and parasites.
