The Immunologist Toolbox Reports

Published Date: 02 Nov 2017

Amanda Fultz

Immunology

Immunologist Toolbox Reports

Haptens

A hapten is a small molecule that, on its own, cannot trigger an immune response and therefore no antibodies against the hapten are produced. However, when this hapten molecule is attached to a carrier protein (usually which also cannot cause an immune response on its own) it is able to trigger an immune response and antihapten antibodies are formed. Once the antibodies have been formed aginst the hapten molecule, the hapten may be able to bind to the antihapten antibodies. However, this itself won’t trigger the immune response, as the carrier protein is still needed for that. Sometimes this binding of just the hapten to the antihapten antibody can actually block the body’s immune response to the hapten-carrier complex, because the complex is unable to bind to the antihapten antibody. This is known as hapten inhibition.

There are several examples of haptens, one of which being the toxin in poison ivy, urushiol. This molecule is not initially a hapten; it is oxidized once it is absorbed into the skin and the oxidized form is the hapten molecule known as quinone. In this case, skin proteins are used as the carrier protein for the hapten molecule. There are also haptens which can cause autoimmune conditions, such as hydralazine. This is a drug that works to lower blood pressure; however, in some cases it can cause lupus erythematosus. Other drugs that can act as haptens and cause drug-induced disease are the anesthetic gas halothane which can lead to hepatitis, and penicillin which can cause hemolytic anemia.

Routes of Immunization and Effects of Antigen Dose

There are many ways to immunize someone against a certain antigen. Common routes include subcutaneous delivery, intradermal or intramuscular injection. While most people are familiar with the traditional subcutaneous delivery of the vaccine into your arm (which does generally confer the strongest response), there are also non-parenteral options such as mucosal or transcutaneous, as well as parenteral delivery in different areas of the body. There are advantages to mucosally or transcutaneously administered vaccines over the traditional vaccines that include the potential to generate not only systemic immunity but mucosal immunity, the fact that they’re a potentially more stable vaccine, an increased shelf life, and also the fact that vaccines administered without the use of needles allows for people besides specially trained healthcare members to give the vaccine (especially useful for vaccination in third world countries or areas in need of widespread immunization). A limitation in developing these alternatively administered vaccines is that safe, effective adjuvents must be developed.

The above diagrams are from an experiment where mice were immunized and titer levels were measured. In the first graph, the comparison is between three different routes of immunization: subcutaneously at the base of the tail, intraperitoneal, and intraplantar in a hind footpad. As demonstrated by the data in the graph, the load and slope were similar in all three routes, and the subcutaneous base of tail immunization and the intraplantar footpad route were similar in their titer. The intraperitoneal route was believed to not be as effective because of shortened survival of the antigen depot (see adjuvants). From this experiment it can be concluded that the route of immunization is in fact important in conferring immunity.

In the second graph, the comparison is between mice injected twice with the vaccine and hose injected four times with the vaccine. The change in titer over time is very consistent between the two groups. What is apparent from this data is that the higher number of subcutaneous injections given, the higher the antibody titers. At too low of a dose, there is a possibility of no immune response; too high of a dose and the immune response can actually be inhibited.

Adjuvants

Adjuvants are important components of vaccines. They are either a chemical or biological molecule that is linked to the antigen in question (whatever the vaccine is for), and they work to cause the immune system to produce antibodies and/or trigger cell-mediated immunity against the antigen the adjuvant is attached to. The adjuvant itself is NOT antigenic. The T helper cell response is greatly important in a vaccine’s efficacy, and adjuvants can greatly impact the T helper cell response. For a long time, the only adjuvants used in vaccines for human use have been aluminum salts. With recent increased knowledge of the mechanism of adjuvents, new ones have been created. This diagram shows the adjuvant enhancing the adaptive immune response, which involves B cells and T cells. B cells are activated and differentiate into either memory B cells or antibody-secreting plasma cells. Th1 cells activate macrophages by secretion of IFN-g, and this process causes B cells to produce antibodies that will opsonize the antigen. Cytotoxic T cells and natural killer cells are also activated. The Th2 cells cause B cells to make antibodies that neutralize the antigen (as opposed to opsonize) by secreting cytokines like interleukin-4.

There are a few different kinds of adjuvants, and they each have different mechanisms of how they work. Alum (which is used in the DTP, human papillomavirus, and hepatitis vaccines) and emulsions (IFA and MF59) generate depots that trap antigens where the vaccine is injected. This enables a slow release of the antigens, leading to a constant stimulation of the immune system. IFA and MF59 emulsion adjuvants can also induce MHC responses. Some adjuvants induce innate immunity by acting as ligands for pattern recognition receptors (this includes toll-like receptors). They also affect the adaptive immunity by targeting antigen presenting cells. Interestingly, LPS from gram negative bacteria has the potential to be an adjuvant because it is a ligand for TLR4, however it is much too pyrogenic to be used in vaccines. A synthetic derivative (MPLA) has been created and is used clinically in Europe.

Affinity chromatography

Affinity chromatography is a laboratory technique used to separate a mixture by making use of a very specific interaction, such as the one between an enzyme and substrate, a receptor and ligand, or more frequently in an immunologists’ case, between an antigen and antibody. Affinity chromatography is often used to purify nucleic acids, purify proteins from cell free extracts, and the purification of antibodies from serum. To purify antibody from serum, the column method is often used. The antigen for which the desired antibody is specific for is bound to small chemically reactive beads. This is then loaded into the column, while the antiserum is poured in and allowed to pass over the beads with the bound antigen. The antibodies that are specific for the antigen will bind, and anything else in the serum will pass through and be discarded. The desired antibodies are released by dramatically lowering or raising the pH. Other techniques are available based on the desired molecule being isolated (protein, nucleic acid, etc.).

Equilibrium dialysis: measurement of antibody affinity and avidity

Equilibrium dialysis is a technique that can be used to determine the affinity of an antibody. To set it up, you obtain a dialysis membrane and create a bag. In this bag you place a known concentration of an antibody that is unable to diffuse through the membrane due to their large size, as well as various antigens. Some antigens will be bound by the antibodies in the bag and thus will also be able to diffuse through the membrane. The concentration of antigen inside and outside the dialysis bag can be measure and compared; this data, plus the knowledge of the amount of antibody present, can help determine he affinity of the antibody. This is demonstrated by the first diagram. The second diagram shows the Scatchard analysis, which is how the data can be analyzed to determine number of binding sites (number of moles of antigen bound per mole of antibody).

Avidity is the strength of the binding between the antibody and antigen. Sometimes an antigen that has several of the exact same epitope will be bound by both binding sites of the antibody, thus increasing the avidity (i.e. IgM has ten binding sites, so avidity has the potential to be extremely high).

Coombs tests and the detection of Rhesus incompatibility

Monoclonal antibodies and Phage display libraries for antibody V-region production

Immunofluorescence microscopy

Immunoprecipitation and Immunoblotting

Use of antibodies in the isolation and identification of genes and their products

Flow cytometry and FACS analysis

Biosensor assays for measuring rates of associates and disassociation of antigen receptors for their ligands

Stimulation of lymphocyte proliferation by treatment with polyclonal mitogens

Measurements or apoptosis by the TUNEL assay

Assays for cytotoxic T cells and CD4 T cells

DNA Microarrays

Assessment and transfer of protective immunity

Testing for allergic responses

Hematopoietic cell transfers

In vivo depletion of T cells and B cells

Transgenic and Knockout animals