The word affinity is used to describe the strength of binding between one antibody binding site
and an antigenic determinant (epitope or hapten). The association constant is the mathematical
value that is the measure of the strength of binding.
Anibody molecules are multivalent and antigens are also often multivalent. This multivalency
tends to increase the strength of the interaction, and this really represents the true state of affairs.
This overall binding energy that results in the binding of a multivalent antibody with a
multivalent antigen is called the functional affinity or the avidity.
Agglutination Reactions
The discussion covering zeta Potential and the Coombs Test (which is also known as the antiglobulin test or the anti-immunoglobulin test) is very important. What is the distinction
between the direct Coombs test and the indirect Coombs test?
Passive agglutination tests involve attaching an antigen to a particle of some sort (latex beads,
tanned red blood cells) and then running an agglutination reaction with antibody.
Precipitation Reactions
This describes the reaction between soluble antibody and soluble antigen in which an insoluble
product results. (Remember that precipitation is a secondary phenomenon. Ag-Ab reactions
may occur and form soluble immune complexes even without the production of a visible
Precipitation reactions can be done in a variety of ways:
In test tubes
In agarose gels:
As double diffusion
As single diffusion
After electrophoresis
Solid phase immunoassays;
ELISA – Enzyme linked immunosorbant assay
You need reagent antibodies or reagent binding proteins that have been
“tagged” with an enzyme label. This means that the enzyme has been
covalently coupled to the protein reagent.
a. Typical enzymes include: Horseradish peroxidase or Alkaline
b. Reagent proteins include antibodies such as goat-anti-human-IgG. Or,
bacterial proteins that bind to antibodies such as Staphylococcal
protein A or Streptococcal protein G. Biotin and Avidin can also be
used ---- avidin has several high-affinity binding sites for biotin thus it
can be used to bridge molecules that have been “tagged” with biotin.
2. You need some kind of “solid phase” to which proteins can stick. This would
usually be some kind of plastic microtiter plate. Thus, as is the example in
Fig 5.11, antigen can be used to “naturally” coat the wells of a microtiter
plate. After you do this you would have to “block” the plate with some kind
of irrelevant protein (or detergent). We usually use a milk solution for this
that is called BLOTTO.
3. One then reacts the antigen-coated plate with appropriate enzyme-tagged
antibody. (You can actually sandwich several different reagents at this point –
as will be discussed in class.) Afterwards the appropriate substrate is added
and a color-change will indicate a “positive” test.
Western Blot
In these assays, antigens are first electrophoresed and then blotted onto a piece of
filter-paper. Thus the filter paper is the solid phase to which the antigen is
The filter is blocked with BLOTTO and then reacted with appropriate “tagged”
antibodies and then substrate. As above, a color reaction indicates a “positive”
In these assays, antibodies or other reagent proteins are “tagged” or labeled with fluorescent
dyes. These fluorescent reagents can then be used to stain samples mounted onto microscope
slides and the slides can be examined using a fluorescent microscope.
Typical fluorescent labels include FITC, TRITC, PE and many others.
Cells in suspension can also be stained fluorescently and then analyzed by fluorescenceactivated cell sorting or FACS analyis.
Reagent Antibodies
For many of the serological tests described above, it is essential to have antibodies of defined
specificity which can be used as reagents in the tests.
Traditionally such antibodies were made as polyclonal antibodies. To make such antibodies,
very pure antigen was injected into subject animals and then after an appropriate amount of time
(often after several “booster shots”) the antibody containing serum was harvested from the
animal by bleeding.
Such polyclonal antisera are very valuable, but they do have some limitations.
Particularly, such antisera contain populations of antibodies that react to all the
epitopes of the antigen prep – including any impurities that might have
contaminated the antigen. Of course, polyclonal antisera also contain multiple
isotypes of antibody too.
To get around this problem, one can immunize an animal and then remove and culture and clone
its B-cells. In this technique, one then cultures single clones of B-cells, each clone producing
only one specificity of antibody, reactive with only one epitope of the immunizing antigen.
This procedure involves a number of sophisticated tricks that we will list here and
discuss in more detail in class.
1. Normal B-cells will not survive very long in culture so one must immortalize
them by giving them genes from cancerous B-cells.
 Cancerous plasma cells (antibody secreting B-cells) are called
myeloma cells.
 B-cells from immunized animals are mixed with myeloma cells in the
presence of a mild detergent (polyethylene glycol or PEG). This
causes cells to fuse forming hybridomas or hybrids between normal
and myeloma cells.
2. A selection system is used that preferentially allows the hybridoma cells to
grow while suppressing the growth of any non-hybridized myeloma cells.
3. Hybridoma cells are fragile, so feeder cells of macrophages are used to
produce growth promoting cytokines.
4. Appropriate immunoassays (usually ELISA’s) have that allow you detect any
antibodies being produced by the hybridoma clones.
Related flashcards
Blood proteins

22 Cards

Protein complexes

25 Cards

Protein structure

38 Cards

Create flashcards