Antigen Binding

1.            Immunization

a.            Immunization is the deliberate induction of an immune response.

b.         To determine whether an immune response has occurred and to follow its course, the immunized individual is monitored for the appearance of immune reactants directed at the specific antigen.

c.         Immune responses to most antigens elicit the production of both specific antibodies and specific effector T cells.

d.            Monitoring the antibody response involves analysis of relatively crude preparations of antiserum.

e.         This is the fluid phase of clotted blood which, in an immunized individual, is called antiserum because it contains specific antibodies against the immunizing antigen as well as other soluble serum proteins.

2.            Immunogens and Haptens

a.         An immunogen is any substance that induce an immune response.

b.         An antigen is a molecule that binds to a specific antibody.

c.         All immunogens are antigens, not all antigens are immunogenic – some need to be attached to an immunogen in order to elicit antibodies.

d.            Haptens:

i.          a molecule that is not immunogenic by itself but can react with specific antibody.

ii.          they are usually small molecules and some high molecular weight nuclear acids.

e.            Haptens are not immunogenic because they cannot activate helper T cells.

f.          The failure of haptens to activate is due to their inability to bind to MHC proteins.

g.            Activation of Haptens:

i.            although haptens cannot stimulate a primary or secondary response by themselves, they can do so when covalently bound to a carrier protein.

ii.          in this process, the hapten interacts with an IgM receptor on the B cell and the hapten-carrier protein complex is internalized.

iii.         a peptide of the carrier protein is presented in association with class II MHC protein to the T helper cells.

iv.         the activated helper T cell then produces interleukins, which stimulate the B cells to produce antibody to the hapten.

3.         Cross-reactivity

a.         A cross reaction is the binding of an antibody to an antigen other than the immunogen.

b.         Most cross-react with closely related molecules but some are specific for antigens having no clear relationship to the immunogen.

c.         These cross-reacting antibodies can create problems when the antiserum is used for detection of specific antigen.

d.         They can be removed from an antiserum by absorption with the cross-reactive antigen, leaving behind the antibodies that bind only to the immunogen.

e.         This can be performed using immobilized antigen by affinity chromatography, which is also used for purification of antibodies or antigens.

f.          The problems resulting from the heterogeneity of the antibodies present in an antiserum can be avoided by making monoclonal antibodies, which are homogenous antibodies derived from a single antibody-producing cell.

g.         These can be selected for lack of cross-reactivity and their binding properties can be defined more reliably.

4.            Immunogenicity

a.         There are many factors affecting the ability of a foreign molecule to elicit an immune response.

b.         Type of molecules:

i.            although any structure can be recognized by antibody as an antigen, usually only proteins elicit fully developed adaptive immune responses.

ii.          proteins engage T cells because the T cells recognize antigens as peptide fragments of proteins bound to MHC.

c.            Molecular size:

i.          the most potent immunogens are proteins with high molecular weight, i.e. above 100,000.

ii.            molecules with molecular weight below 10,000 are weakly immunogenic.

iii.         the larger and more complex a protein, and the more distant its relationship to self proteins, the more likely it is to elicit a response.

iv.         this is because such responses depend on the protein being degraded into peptides that can bind to MHC molecules, and on subsequent recognition of these peptides.

v.            particulate or aggregated antigens are more immunogenic because they are taken up more efficiently by the specialized antigen-presenting cells responsible for initiating a response.

d.            Antigenic determinants (epitopes):

i.            epitopes are small chemical groups on the antigen molecule that can elicit and react with antibody.

ii.          an antigen can have one or more determinants.

e.            Dosage:

i.          below a certain threshold dose, most proteins do not elicit an immune response.

ii.          above the threshold dose, there is a gradual increase in the response with increasing dose to a broad plateau level, followed by a decline at very high antigen doses.

iii.         as most infectious agents enter the body in small numbers, immune responses are generally elicited only by pathogens that multiply to a level sufficient to exceed the antigen dose threshold.

iv.         the broad-response optimum allows the system to respond to infectious agents across a wide range of doses.

v.         at very high antigen doses the immune response is inhibited, which may be important in maintaining tolerance to abundant self proteins.

f.          Route of administration:

i.          antigens injected subcutaneously generally elicit the strongest responses.

ii.          antigens injected or transfused directly into the bloodstream that are readily taken up by antigen-presenting cells, tend to induce unresponsiveness unless they bind to host cells.

iii.         antigens administered solely to the gastrointestinal tract have distinctive effects, eliciting a local antibody response in the intestinal lamina propria while producing a state of systemic tolerance.

iv.            important in avoiding allergy to antigens in food while systemic inhibition of immunity prevent formation of IgE antibodies, which are the causes of such allergies.

g.            Foreignness:

i.          in general, molecules recognized as ‘self’ are not immunogenic.

ii.          to be immunogenic, molecules must be recognized as non-self or foreign.

h.            Chemical-structural complexity:

i.          a certain amount of structural or chemical complexity is required.

ii.          amino acids homopolymers are less immunogenic than heteropolymers containing 2 or 3 different amino acids.

5.            Antigen Presentation to T and B cells

a.         B cells, with their membrane-bound antibody, recognize and bind free antigen in solution.

b.         The epitopes on the antigen must be on the ‘outside’ of the molecule, accessible for interaction with the receptor.

c.            Terminal side chains of polysaccharides and hydrophilic portions on protein molecules generally constitute B cell epitopes.

d.         The interaction of epitope with T-cell receptors requires:

i.          prior processing of the antigen and

ii.          the association with an area of the processed antigen with Major Histocompatibility Complex (MHC) molecules present on the surface of antigen-presenting cells.

e.            Generally such ‘processed’ epitopes are internal denatured linear hydrophobic areas of proteins.

f.            Polysaccharides do not yield such areas and indeed are not known to bind or activate T cells; in contrast, such areas are obtained following processing of proteins.

g.         Thus polysaccharides contain solely B cell recognizable epitopes whereas proteins contain both B and T cell recognizable epitopes.

6.         Value of Vaccines

a.            Vaccines aim to protect an individual to an infectious agent prior to possible exposure by exposing the body to an attenuated form of the agent so that resistance could be generated.

b.            Protection through passive immunization is the objective achieved by injections of immune globulin to protect an individual against the infective agent.

c.            Protection against development of disease can also be afforded by post-exposure immunization if the latent period of the disease is long, e.g. administration of rabies vaccine against rabies virus.

d.         There is potential for use of vaccines to prevent certain cancers in humans, e.g. use of HBV vaccine in high-risk groups may provide protection against development of hepatocellular carcinoma.

7.            Adjuvants

a.         An adjuvant is any substance that enhances the immunogenicity of substances mixed with it.

b.         Most proteins are poorly immunogenic or non-immunogenic when administered by themselves – strong adaptive immune responses to protein antigens almost always require that the antigen be injected in a mixture of adjuvant.

c.            Adjuvants differ from protein carriers in that they do not form stable linkages with the immunogens.

d.            Mechanism of enhancing immunogenicity:

i.            adjuvants convert soluble protein antigens into particulate material, which is more readily ingested by antigen-presenting cells, such as macrophages.

ii.          the antigen can be adsorbed on particles of the adjuvant (such as alum) or made particulate by emulsification in mineral oils.

iii.         some adjuvants contain bacteria or bacterial products which enhance immunogenicity.

e.         Some human vaccines contain adjuvants such as aluminum hydroxide or lipids.

f.            Adjuvants that enhance immune responses: